Distinction between glycoprotein IIIa and the 100-kDa membrane protein (aggregin) mediating ADP-induced platelet activation

Previous studies from our laboratories showed that 5'-p-fluorosulfonylbenzoyl adenosine (FSBA) inhibits ADP-induced platelet shape change, aggregation, and exposure of fibrinogen sites while covalently binding to 100-kDa platelet membrane protein (aggregin) on the intact platelet. Chymotrypsin digests aggregin to a fragment of 70 kDa, abolishing the inhibition, and also cleaves platelet glycoprotein IIIa (GPIIIa) (100 kDa) to a 70-kDa fragment containing the P1A1 epitope. We questioned whether these platelet membrane proteins were distinct. Both 5'-p-[3H]sulfonylbenzoyl adenosine (SBA)-labeled aggregin and 125I-GPIIIa were precipitated by polyclonal antibodies to a 100-kDa fraction of platelet membranes, but aggregin was not precipitated by a monospecific antibody to P1A1 which precipitates GPIIIa. Further a monospecific polyclonal antibody to immunopurified GPIIIa coupled to protein A-Sepharose adsorbed GPIIIa but not aggregin. Similarly, both aggregin and GPIIIa were precipitated by a polyclonal antibody to an isolated 70-kDa component of platelet membrane but only GPIIIa was precipitated by the monoclonal antibody to GPIIIa, (SSA6). Two patients with Glanzman's thrombasthenia whose platelet membranes contained less than 5% GPIIIa as assayed by monoclonal antibody binding (A2A6), incorporated [3H]SBA to the same extent as normal individuals. Furthermore, FSBA inhibited ADP-induced shape change with a similar concentration dependence for both thrombasthenic and normal platelets. Finally, mobility of GPIIIa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was decreased following reduction with dithiothreitol whereas that of [3H]SBA-labeled MP 100 was not altered. We conclude that GPIIIa and aggregin are distinct platelet membrane proteins.     

Linear dichroism and orientational studies of carotenoid Langmuir-Blodgett films

The linear dichroism of single monolayers of lutein, zeaxanthin and a mixture of lutein and synthetic phosphatidylcholine has been measured. The angle of orientation of the carotenoid molecules was found to lie between 45° and 51° relative to the plane of the solid support. Although the adsorbed monolayers were mostly in a monomeric state, microscopic observations, as well as the II-A isotherms, indicated the existence of crystalline islets. The results have been interpreted in connection with Haidinger's polarization brushes.     

Mapping of mouse gamma crystallin genes on chromosome 1

Restriction fragments analysis of DNA from mouse-hamster somatic-cell hybrid clones revealed that a mouse gamma crystallin cDNA hybridized to genomic sequences located on mouse chromosome 1. Identification of restriction fragment length polymorphisms (RFLPs) in the gamma crystallin sequences of inbred strains of mice permitted the further localization of the gamma crystallin genes (Cryg) to the proximal region of chromosome 1 closely linked to the loci encoding isocitrate dehydrogenase (Idh-1), a low molecular weight (LM) crystallin protein polymorphism (Len-1), and fibronectin (Fn-1). A single recombinant was observed betweenLen-1 and an RFLP in the gamma crystallin gene family, consistent with the hypothesis thatLen-1 is one of the several structural loci encoding gamma crystallin genes.Len-1 is probably located on the centromeric end of theCryg gene family. Linkage ofIdh-1, Cryg, andFn-1 in mice extends the syntenic relationship of those loci to the human, bovine, and rodent genomes and may define a chromosomal region that is generally conserved among mammals. The map position ofCryg, near the eye lens obsolescence (Elo) locus, was confirmed by the discovery that the restriction fragment patterns of gamma crystallin sequences differed between strain C3H/HeJ and the congenic anophthalmic mutant strain, C3H.Elo. Therefore, the gamma crystallin genes were contransferred with the mutantElo gene in the derivation of C3H.Elo. The results establish that LEN-1 is a marker for the gamma crystallin gene family, position the gamma crystallin gene family relative to other markers on mouse chromosome 1, and provide additional evidence that theElo mutation is encoded at a locus closely linked to the gamma crystallin gene cluster. This study found no evidence of recombination hot spots within the gamma crystallin gene cluster.     

The interaction of quinone analogues with wild-type and ubiquinone-deficient yeast mitochondria

The interaction of the exogenous quinones, duroquinone (DQ) and the decyl analogue of ubiquinone (DB) with the mitochondrial respiratory chain was studied in both wild-type and a ubiquinone-deficient mutant of yeast. DQ can be reduced directly by NADH dehydrogenase, but cannot be reduced by succinate dehydrogenase in the absence of endogenous ubiquinone. The succinate-driven reduction of DQ can be stimulated by DB in a reaction inhibited 50% by antimycin and 70-80% by the combined use of antimycin and myxothiazol, suggesting that electron transfer occurs via the cytochrome b-c1 complex. Both DQ and DB can effectively mediate the reduction of cytochrome b by the primary dehydrogenases through center o, but their ability to mediate the reduction of cytochrome b through center i is negligible. Two reaction sites for ubiquinol seem to be present at center o: one is independent of endogenous Q6 with a high reaction rate and a high Km; the other is affected by endogenous Q6 and has a low reaction rate and a low Km. By contrast, only one ubiquinol reaction site was observed at center i, where DB appears to compete with endogenous Q6. DB can oxidize most of the pre-reduced cytochrome b, while DQ can oxidize only 50%. On the basis of these data, the possible binding patterns of DB on different Q-reaction sites and the requirement for ubiquinone in the continuous oxidation of DQH are discussed.     

α-Helix-to-random-coil transitions of two-chain coiled coils: Experiments on the thermal denaturation of ββ tropomyosin cross-linked selectively at C-190

A method is described for preparation of a species of β tropomyosin that is sulfhydryl-blocked at C36 and disulfide-cross-linked at C190. Five steps are involved: (1) Rabbit skeletal muscle tropomyosin, comprising αα and αβ species, is oxidized with ferricyanide, disulfide-cross-linking both species at C190. (2) The product is treated with iodoacetamide, blocking the only remaining free sulfhydryl, i.e., C36 of the β-chains. (3) The C36-blocked, C190-cross-linked product is reduced with dithiothreitol (DTT), unfolded in urea, and α and β chains separated by ion-exchange chromatography. (4) The C36-blocked β chains are refolded by dialysis. (5) The refolded, C36-blocked ββ species are cross-linked at C190 by ferricyanide oxidation. The resulting C36-blocked, C190-cross-linked ββ product is separated from contaminating species—mostly completely blocked β-chains and multichain cross-linked molecules—by size-exclusion chromatography in denaturing (guanidinium chloride) solvent. The five-step process and the final product were monitored by titration of free sulfhydryls and by NaDodSO₄/polyacrylamide gel electrophoresis (PAGE). Thermal unfolding curves from CD are reported for the resulting pure, C36-blocked, C190-cross-linked ββ species and for its DTT-reduction product, the noncross-linked C36-blocked species. The latter shows almost the same thermal unfolding transition as intact, noncross-linked ββ species. The former shows a pretransition similar to, but larger in extent than, the one well known to occur in the analogous case of C190-cross-linked αα tropomyosin. These unfolding transitions are compared with one another and with that previously reported for doubly cross-linked (at C36 and C190) ββ species. These comparisons are made in the light of current physical models for coiled-coil unfolding equilibria. It is concluded that although no extent model is demonstrably satisfactory, any successful model must include strain at the cross-link, loop entropy, and regional nonuniformities as essential parts of the physics.     

Characterization of rat Leydig cell gonadotropin receptor structure by affinity cross-linking

The present study was intended to examine the structure of the rat Leydig cell gonadotropin receptor. Leydig cell suspensions were prepared by either collagenase digestion or mechanical disruption of the testes. The cells were incubated with 125I-human chorionic gonadotropin (hCG) following which the bound 125I-hCG was covalently cross-linked to the cell surface receptor using a cleavable (dithiobis(succinimidyl propionate] and a noncleavable (disuccinimidyl suberate) cross-linking reagent. The extracted cross-linked membrane proteins were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions and subjected to autoradiographic analysis. Under nonreducing conditions, three radiolabeled bands, in addition to intact hCG and its alpha-subunit, were detected with apparent molecular weights of 184,000, 136,000, and 103,000. However, under reducing conditions, three radiolabeled bands migrated on the gel corresponding to molecular weights of 144,000, 106,000, and 75,000. The binding of 125I-hCG to the receptor was inhibited by hCG and luteinizing hormone, but not by a number of other peptides or proteins. The radiolabeled bands were not detectable in hCG down-regulated Leydig cells. Furthermore, a similar autoradiographic pattern of 125I-hCG-linked complexes was seen when the 125I-linked receptor complex was subjected to immunoprecipitation with anti-hCG antibodies followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition, evidence was obtained indicating that these three labeled bands were derived from the same molecular species. The data suggests that the hCG receptor in Leydig cell is probably an oligomeric complex with a molecular weight of about 250,000, which is composed of three polypeptide chains of molecular weights 121,000, 83,000, and 52,000 held together through noncovalent forces. Additionally, collagenase treatment of Leydig cells does not appear to alter the autoradiographic pattern of the 125I-hCG-linked receptor.     

Testing the two-state model: anomalous effector binding to human hemoglobin

Three allosteric states are required to describe the relaxation of (carbon monoxy) hemoglobin following flash photolysis. Combined absorbance and fluorescence probes were used. The absorbance signals consist of a component corresponding to ligand recombination and a component for the R-T transition. The fluorescence of 8-hydroxy-1,3,6-pyrenetrisulfonate (HPT), an analogue of 2,3-diphosphoglycerate, shows rates and amplitudes correlated with the absorbance transients. Measurements were made at pII 6, 6.5, and 7.0 at CO partial pressures of 0.1 and 1 atm. The fractional photolysis was varied in each case to change the initial distribution of the R states. Data show an immediate absorbance change due to ligand dissociation, while the changes in the ligand isosbestic and the fluorescence signals occur with time constants of 80 microseconds (at pH 6.5). The signals then show a biphasic return to equilibrium, characteristic of the allosteric system. The measurements provide three independent probes of the kinetics of the substates of hemoglobin. Although the ligand binding data can be generally represented by a two-state model, the fluorescence data require T states with different affinities for HPT.     

Topographic modeling of free and methionyl-tRNA synthetase bound tRNAfMet by singlet-singlet energy transfer: bending of the 3'-terminal arm in tRNAfMet

Conformations of tRNAfMet, free and methionyl-tRNA synthetase bound forms, are analyzed by using singlet-singlet energy transfer as a spectroscopic ruler. tRNAfMet(8-13,3'-Flc), tRNAfMet(8-13,D-Etd), and tRNAfMet(3'-Flc,D-Etd) are prepared by sequential chemical modifications. The methionyl-tRNA synthetase binding affinity of these double-labeled tRNAfMets is similar to those of unmodified tRNAfMet. The fluorescence properties of the individual fluorophore in these tRNAs, including emission spectra, anisotropy, and quenching by methionyl-tRNA synthetase, are similar to those of single-labeled tRNAfMet. The transfer efficiencies of double-labeled tRNAfMets, as determined by both donor quenching and sensitized emission, showed efficient energy transfer in all cases. Random orientation being assumed, the apparent distances are 25 A between 8-13 and D20, 44 A between 8-13 and the 3'-terminus, and 49 A between the 3'-terminus and D20, respectively, in free tRNAfMet. Upon binding of methionyl-tRNA synthetase, the apparent distances are 25 A between 8-13 and D20, 45 A between 8-13 and the 3'-terminus, and 54 A between the 3'-terminus and D20, respectively. These results provide topographic models of these specific locations in free and methionyl-tRNA synthetase bound tRNAfMet and suggest that the immobilized 3'-terminal arm in the amino acid acceptor stem bends toward the inner loop of the L-shaped tRNA upon binding of methionyl-tRNA synthetase.     

Studies on the phospholipid composition of pathogenic cell membranes of Mycoplasma hyopneumoniae

The membrane phospholipid and fatty acid compositions of Mycoplasma hyopneumoniae, a pathogen of porcine enzootic pneumoniae isolated in China, was studied by thin-layer chromatography and gas chromatography. The results showed that membrane phospholipids consisted predominantly of diphosphatidylglycerol. The percentage of C16 - C18 fatty acids comprised 79% of the total fatty acids, of which oleic acid as well as palmitic acid are the major fatty acids. Some differences were shown in fatty acid composition as compared with membranes of other species of Mycoplasma.     

Genealogy of Principal Strains of the Yeast Genetic Stock Center

We have constructed a genealogy of strain S288C, from which many of the mutant and segregant strains currently used in studies on the genetics and molecular biology of Saccharomyces cerevisiae have been derived. We have determined that its six progenitor strains were EM93, EM126, NRRL YB-210 and the three baking strains Yeast Foam, FLD and LK. We have estimated that approximately 88% of the gene pool of S288C is contributed by strain EM93. The principal ancestral genotypes were those of segregant strains EM93-1C and EM93-3B, initially distributed by C. C. Lindegren to several laboratories. We have analyzed an isolate of a lyophilized culture of strain EM93 and determined its genotype as MATa/MATα SUC2/SUC2 GAL2/gal2 MAL/MAL mel/mel CUP1/cup1 FLO1/flo1. Strain EM93 is therefore the probable origin of genes SUC2, gal2, CUP1 and flo1 of S288C. We give details of the current availability of several of the progenitor strains and propose that this genealogy should be of assistance in elucidating the origins of several types of genetic and molecular heterogeneities in Saccharomyces.